A subset of genes in mammals is regulated by genomic imprinting, a process that results in unequal expression of the maternal and paternal alleles of this class of genes. The existence of imprinted genes is hypothesized to explain why nuclear contributions from both parents are required for normal mammalian development. Furthermore, imprinting plays a role in the transmission of a number of human disorders, including Beckwith-Wiedemann Syndrome (BWS), Silver-Russell Syndrome (SRS), Prader-Willi Syndrome and Angelman Syndrome, in that the sex of the parent that transmits the affected gene(s) determines whether offspring will be affected. Aberrant imprinted gene expression is also involved in the onset or progression of cancers, such as Wilms tumors. The overall goal of our work is to elucidate the mechanism by which parental identity of imprinted genes is established and maintained. The studies will employ the conserved H19 and Igf2 locus. The imprinting of H19, which produces a non-coding RNA from the maternally-derived allele, and the linked and oppositely imprinted growth-promoting Igf2 gene is mediated through the 2 kb imprinting control region (ICR) and shared enhancers. The ICR, which is also designated the differentially methylated domain (DMD), acts as a methylation-senstitive, CTCF-dependent insulator. When unmethylated on the maternal allele, the insulator allows H19 exclusive access to shared enhancers. In contrast, a methylated paternal insulator enables Igf2 to engage the enhancers. This proposal will investigate the mechanism of H19/Igf2 imprinting through the following experiments. Individuals with BWS, sporadic Wilms tumors and SRS have been identified that have microdeletions and epimutations (hypermethylation) in the human ICR and aberrant imprinted regulation of H19 and Igf2. Specific Aim 1 will generate mouse mutants and human iPS cells to elucidate the mechanisms governing the loss of imprinted gene regulation in these individuals. DNA hypermethylation of the paternal ICR allele is associated with repression of the H19 gene, but the precise sequences and mechanism mediating repression remain incompletely defined. We have shown that mutation of CpGs within the CTCF binding sites on the paternal allele causes demethylation and ectopic insulator function, whereas a similar number of CpG mutations outside of the CTCF binding sites but within the ICR has minimal effects. We propose that the CpG dinucleotides within the CTCF binding sites are critical for paternal- specific repression. In Specific Aim 2, we will test this hypothesis by mutating all CpG outside of the CTCF binding sites but within the ICR and assessing function in vitro by repression assays and in vivo through gene targeting experiments. Finally, a novel highly expressed imprinted non-coding RNA has been identified between the H19 and Igf2 genes. Specific Aim 3 will assess the role of this RNA by in vitro assays and in vivo gene targeting experiments. Together these experiments will allow a greater understanding of imprinted gene regulation at this important locus as well as serving for a model of epigenetic gene regulation in mammals. .